The molecular biological approach to the study of the mitochondrial genetic code and the regulation of expression of mitochondrial genes will include the following specific investigations directed at the yeast genes oxi-1 and oxi-2, which code for cytochrome oxidase subunits II and III: a) DNA sequence analysis of mutations in these genes, already in hand, including those that cause premature polypeptide chain termination in vivo. Mutations generating codons not normally used in yeast mitochondrial genes may be identified. The information will be further applied to the further study of suppressor mutations in the mitochondria. b) The isolation and characterization of spontaneous mutations in these mitochondrial genes. This information will have evolutionary significance and eventually serve as a basis for the detailed study of specific effects of mutagens on mitochondrial DNA. c) Development of a system for the genetic transformation of yeast mitochondria to study the movement of genes within the cell. d) Detailed characterization of the transcripts copied from the oxi-1 and oxi-2 genes, both in wild-type and mutant strains. The effect of changes in the physiological growth conditions on mitochondrial transcription will also be examined. e) A study of the mechanisms by which three previously described nuclear genes regulate the expression of the oxi-1 and oxi-2 genes, by examining their effects on mitochondrial transcription and by cloning the nuclear genes themselves. These studies will bear on the evolution of organelle systems in eucaryotic cells, and on the intracellular coordination of distinct genetic systems. These questions can be studied in the greatest depth with the well developed genetic system of yeast.